In developing new substances and developing new uses of substances, it is important to verify the effects of the substances on the human body depending on the use form thereof. Among these, in developing pharmaceuticals, food additives, and the like, it is essential to verify what kind of effects these substances have on the human body.
For such verification, animal experiments and experiments at a cell level using cultured cells are generally carried out, but in accordance with technological advances, higher-throughput cell-level assays using cultured cells have been required.
On the other hand, in monolayer culture, which is generally used in a cell assay, the environment around the cells is greatly different from the environment in the animal body, and therefore there is a problem in that most functions which are expressed in the body are lost in such cultured cells.
In next-generation cell assay technologies, a tissue mimicking a three-dimensional structure in vivo is artificially reconstructed and a more reliable assay using tissue having a higher level of function is employed, and therefore, it is expected that a higher level of a vivo-vitro correlation can be obtained.
As a cell culture method, there is a method using hydrogel. A hydrogel has excellent characteristics as a carrier of cells such as high-water content, ease of adjusting mechanical properties, excellent nutrient diffuseness, and the like. An embedment culture method in which cells are cultured in a state where the cells are three-dimensionally dispersed inside a hydrogel is known as a method for enhancing the function of cells by interaction between cells and the hydrogel.
In the embedment culture method, it is known that a molecular structure of a polymer constituting a hydrogel greatly influences the function of cells. Particularly, many examples are known in which cells exhibit a high level of function in a hydrogel having, as a main component, a biological polymer including an extracellular matrix such as collagen, gelatin, laminin, and Matrigel.
It is known that collagen exists in various tissues in a living body and is a main component of an extracellular matrix, and it is known that many hydrogels containing collagen as a main component have cell adhesiveness. Cells that are likely to lose the function in a normal culture dish can maintain the function thereof by applying a collagen gel embedment culture method or a collagen gel sandwich culture method. Gelatin is a polymer of which water solubility increases by modifying collagen with acid, alkali, heat, and the like, and maintains cell adhesiveness while being modified, and therefore is used as a substrate for cell culture as same as collagen. Gelatin is inexpensive compared to collagen, and therefore is frequently used for coating of a culture dish. Matrigel is a cell culture substrate sold by Corning Incorporated, and various growth factors such as transforming growth factor (TG), epithelial cell growth factor (EGF), insulin-like growth factor (IGF), and fibroblast cell growth factor (FGF) are contained in addition to an extracellular matrix such as laminin and collagen which are main components thereof.
On the other hand, it is known that cell growth and function do not increase in a hydrogel containing a synthetic polymer such as polyethylene glycol as a main component (NPL 1). It is known that in the hydrogel of a synthetic polymer, arginine-glycine-aspartic acid (RGD) peptide, which is one of the cell adhesion factors of collagen, is modified into a hydrogel in order to impart cell adhesiveness, and therefore cell function can be increased (NPL 2).
A hydrogel can be imparted with photodegradability by incorporating a photodegradable group into the molecule. A photodegradable gel which can be processed by light has been developed using such photodegradability. Examples of a photodegradable gel include a photodegradable gel which contains polyethylene glycol as a main chain and a nitrobenzyl group in the molecule. It is known that the physical properties of a hydrogel formed from a polymeric monomer having such a constitution can be temporally and spatially controlled by light irradiation, and that such photodegradation has no significant cytotoxicity with respect to living cells.
By culturing cells in such a photodegradable gel, cultured cells can be easily extracted from the gel by irradiating the gel with light to degrade the gel. By using the above, for example, only cells at a specific position can be extracted by irradiating only the specific position of the gel with light so that the gel degrades, and therefore, it is possible to separate a specific cell of the cultured cell group (NPL 3).
As a method for preparing a photodegradable gel, a method using radical polymerization is known (NPLs 4 and 5). In a case of using radical polymerization, when polymerization is carried out in the presence of oxygen, a reaction of gelation may be inhibited in some cases due to oxygen. Furthermore, a radical damages cells and physiologically active substances. Since a polymer compound which can be used as a main chain is limited to a polymeric monomer capable of undergoing radical polymerization, use thereof is limited.
The inventors of the present invention have developed a photocleavable crosslinker capable of forming a photodegradable gel by causing a crosslinking reaction only by mixing with a polymer compound without using radical polymerization (PTL 1). However, this gel absorbs a solvent and dissolves, and an intended structure may not be constructed in some cases. Furthermore, the strength of the gel is low, and there is a problem in that production of a complex three-dimensional structure by which a biological structure is reproduced is difficult.
The inventors of the present invention have found that by increasing the number of branches of the photocleavable crosslinker, it is possible to increase the strength of the gel, and by using the crosslinker, it is possible to form a photodegradable gel which has water content and appropriate water solubility as a cell carrier, and also has the strength that enables construction of a complex and fine three-dimensional structure, and therefore have previously filed the patent application (PTLs 2 and 3 and NPL 6).